We propose to study the function and modulation of ion channels in muscle with the long term goal of understanding their role in muscle membrane excitability and in the process of excitation-contraction coupling. In order to study ion channel properties we will use electrophysiological (voltage-and patch-clamp), and biochemical approaches, including reconstitution in planar lipid bilayers as well as in liposomes. Surface and transverse tubule membranes from frog and rat muscle will be isolated in order to study Na+ and Ca2+ -activated K+ channels in planar bilayers. To compare with the bilayer studies, Na+ channels will be also characterized in cut muscle fibers from the frog. Sarcoplasmic reticulum membranes will be isolated to investigate the properties of native Ca2+ release channels. This membrane preparation will be also used to purify the Ca2+ channels which will be incorporated into vesicles and planar bilayers. Their properties will be compared with those of the channels present in native membranes. All the above-mentioned channels will be studied regarding their selectivity, gating, as well as their pharmacological and regulation profile. The purified calcium channel macromolecule will be characterized in terms of ryanodine and InsP3 binding. The characteristics of Ca2+ release from sarcoplasmic reticulum membranes will be studied in vesicles and skinned muscle fibers. Lipid kinases involved in the metabolism of phosphoinositides and the phospholipase C present in the transverse tubule membrane will be characterized. Phosphoinositide metabolism will be investigated in skinned frog muscle livers. Ca2+ -activated K+ channels and other conductances regulated by extracellular transmitters and intracellular modulators will be studied in Drosophila larval muscle, using both wild and mutant types of this fly. The use of Drosophila larval muscle will allow us to gain a deeper understanding of the molecular basis of ion channel modulation in muscle cells.